Based on a mutational search of the mouse IgH locus, we have implicated the presence of two types of V(D)J recombinational control elements that depend on the integrity of the 100kb intergenic region between the germline IgH VH and DH clusters. One suppresses anti-sense transcription from the downstream DH locus, as deletion of the 100kb VH-D region leads to de novo DH anti-sense transcription that produces long non-coding transcripts in developing B and T cells. This increased transcription is associated with greatly increased D to JH joining in thymocytes implicating a positive role for anti-sense transcription in targeting V(D)J recombination. The second VH-D integenic control element, which we term "5'D4KBS", is contained with a 4kb sequence just upstream of D cluster. Remarkably, this element mediates ordered IgH variable region exon assembly in B cells by suppressing VH joining to Ds that have not joined to JHs and mediates lineage-specific joining by suppressing VH to DJH joining in thymocytes. The 5'D4KBS also enhances utilization of VHs located 2MB distant and therefore influences primary antibody repertoires. We have found that the 5'D4KBS functionally employs a CTCF looping/insulator factor binding site, at least for its lineage-specific function. We propose 3 specific aims designed to determine how these elements function in mice and in humans and, based on these studies, to identify additional regulatory elements. Our first aim is to characterize how the 5'D4KBS controls ordered and lineage specific V(D)J recombination and normalizes VH usage. These studies involve an in depth genetic analysis of the functions of individual factor binding sites within 5'D4KBS. In this regard, our preliminary studies implicate sites that bind CTCF, at least in lineage-specific control. We also will assess how assembly of a DJH intermediate during the V(D)J assembly process inactivates apparently suppressive influences of the 5'D4KBS on VH to DJH joining. Our second aim seeks to genetically scan the 100kb VH-D intergenic region to locate elements implicated in controlling transcription of the downstream D and JH segments and to test physiological roles for such transcription. Based on the apparent absence of IgH allelic exclusion control elements from the VH-D intergenic region, we also propose to search for such elements elsewhere in the mouse IgH locus. Our final aim proposes to characterize V(D)J recombination control in mice that have human VH, D, and JH segments in place of the corresponding mouse sequences. These studies will allow us to test the significance of our findings (e.g. the role of VH-D intergenic elements) in the context of whether they are evolutionarily conserved and provide a means to begin an in depth evaluation of the genetic and epigenetic mechanisms involved in generating the human antibody repertoire.) PUBLIC HEALTH RELEVANCE: Unlike most of our genes, antibody genes are assembled from gene segments to allow the generation of B lymphocytes that can produce a vast diversity of different antibodies. Our studies are aimed at discovering how this antibody gene assembly process is carried out and how it is regulated. Knowledge of antibody gene assembly mechanisms will lead to a better understanding of how the diverse sets of antibodies are generated to fight a multitude of different infections and also, of how mistakes in this gene assembly process can predispose to diseases such as immunodeficiency, autoimmunity, and cancer.)